生物谷報道：來自美國麻州大學醫(yī)學院（University of Massachusetts Medical School）神經(jīng)生物學系的研究人員分離得到了兩個光電響應(yīng)（photoresponse）慢終止（slow termination）的非補足（noncomplementary）果蠅突變，這些突變揭示了一種視覺G蛋白耦聯(lián)受體（G proteincoupled receptor,，GPCR）：視紫質(zhì)失活的突變情況,，為研究Ca2+刺激的GPCRs活性調(diào)控提供了重要資料。這一研究成果公布在11月17日《Cell》雜志上,。
領(lǐng)導(dǎo)這一研究的是來自麻州大學醫(yī)學院的李洪生（Hong-Sheng Li,，音譯）博士，他早年畢業(yè)于武漢大學生命科學學院生化與生物物理學系,，在中科院上海腦研究所獲得博士學位,。
該研究中主要針對的是膜受體活性在保護細胞免受興奮毒素（excitotoxicity）毒害的作用。研究人員通過遺傳學和電生理學方法對其分離得到的兩個非補足性果蠅突變進行分析,，發(fā)現(xiàn)這是一種視紫質(zhì)G蛋白耦聯(lián)受體失活缺陷形式,，最后這一突變基因被辨認為鈣調(diào)素結(jié)合轉(zhuǎn)錄激活因子（calmodulinbinding transcription activator ，dCAMTA),。目前已知視紫質(zhì)調(diào)控因子Arr2并沒有介導(dǎo)這個dCAMTA視覺功能過程,，研究人員通過全基因組篩選發(fā)現(xiàn)了5個dCAMTA靶標基因，其中F box基因：dFbxl4的過量表達可以逆轉(zhuǎn)這些突變表型,。研究人員進一步的研究也發(fā)現(xiàn)體內(nèi)dCAMTA通過與Ca2+傳感鈣調(diào)素相互作用可以刺激表達,。這些研究成果都說明了calmodulin/CAMTA/Fbxl4也許在Ca2+刺激GPCRs的活性長時程反饋調(diào)控中起作用，并阻止由于多余Ca2+流入引起的細胞傷害,。
Figure 1. Mutation of dCAMTA Causes Defective Fly Photoresponse
All flies examined are <3 days old.
(A) ERG responses terminated slowly in tes flies. For all ERG traces, event markers represent 5 s orange light pulses, and scale bars are 5 mV. WT = wild-type.
(B) Whole-cell recordings of isolated tes photoreceptor cells revealed a defective termination of light response. The scale bar and light pulse are 200 pA and 500 ms, respectively, for all whole-cell currents.
(C) Annotated genes in chromosome region 45D5-45E1 (top). tes mutations were mapped to this region using deficiency chromosomes and further located distal to the insertion site (arrowhead) of P{EPgy2}EY02897 by male recombination mapping. The actual dCAMTA gene (bottom) occupies virtually the entire mapped region.
(D) Point mutations in tes alleles shown with respect to the functional domains of dCAMTA. * = stop codon.
(E) dCAMTA protein levels in dark-reared mutant flies. Each lane was loaded with four fly heads. rdgA and norpA are mutants for a DAG kinase and PLC, respectively. s.o. = sine oculis.
(F) A WT dCAMTA cDNA rescued the tes phenotype after being expressed in photoreceptors through a trp gene promoter. Both ERG (left) and whole-cell current (right) are shown.
英文原文出處：
Cell, Vol 127, 847-858, 17 November 2006
The Fly CAMTA Transcription Factor Potentiates Deactivation of Rhodopsin, a G Protein-Coupled Light Receptor
Junhai Han, Ping Gong, Keith Reddig, Mirna Mitra, Peiyi Guo, and Hong-Sheng Li
全文下載：
[Summary] [Full Text] [PDF] [Supplemental Data]
相關(guān)背景：
人類細胞在與營養(yǎng)物質(zhì),、毒素、激素甚至光線反應(yīng)時,，必須能夠發(fā)送啟動或關(guān)閉生命過程的信號,。而GPCRs就是這類信號級聯(lián)（如傳遞視覺信號,、攜帶神經(jīng)信息,、
使白血球工具感染并設(shè)置心跳的時間節(jié)律等）的一個關(guān)鍵部分。此外,，GPCR信號
還在多種重要疾病中扮演關(guān)鍵的角色,。因此，GCPR已經(jīng)成為20種最暢銷藥物中的
12種藥物的作用靶標,，這些藥物包括充血性心力衰竭藥物Coreg,、高血壓藥物
Cozaar、乳腺癌藥物Zoladex,、焦慮藥物Buspar,、精神分裂藥物Clozaril。此外，
Zantac和Claritin的作用靶標也是GPCR,。這類藥物每年的銷售總額高達2000億美
元,。
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小知識1：
G Protein-Coupled Receptors: Cells in the human body communicate by releasing ligands (chemical messengers) that bind to receptors – protein molecules embedded in the membranes of receiving cells. The ligand activates the receptor and triggers a cascade of intra-cellular events, in which the message received by the receptor is transmitted to the interior of the cell. This series of biochemical events causes a change in some aspect of the neuronal cell’s behavior. The nature of this change depends upon a number of factors, including the specific ligand and receptor involved in the communication.
GPCRSystems (animation)
Receptors are classified into "superfamilies" based on similarities in their biochemical and structural properties. Of the principal superfamilies of receptors, Synaptic focuses its receptor and drug discovery efforts on the G protein-coupled superfamily of receptors. Each superfamily is subcategorized into "families," based upon the specific ligands with which they interact. Examples of receptor families within the GPCR superfamily are the serotonin, adrenergic, neuropeptide Y and galanin families of receptors. Each member of each family is called a "receptor subtype."
小知識2：
G-Protein-Coupled Receptors
Clifford R. Robinson
My overall research interests include understanding the folding and assembly of protein structures, the relationships between macromolecular structure and function, and the engineering and design of proteins with novel functions and improved properties. In particular, my group is currently investigating the structure, function, folding, and assembly of G-protein-coupled receptors (GPCRs). Our focus is on obtaining novel structural and conformational information, and using protein engineering approaches to produce major advances in the study of GPCR structural biology, biochemistry, and biophysics. We have developed new insights into determinants of structure, stability, and mechanism of signal transduction of this vitally important class of proteins. My team is committed to remaining on the cutting edge of research in biological macromolecules with medically important functions.
GPCR Structural Biology
We are aggressively pursuing structures of several GPCRs, including adenosine A2a and A3 receptors, and neurokinin NK1 and NK2 receptors. We typically express GPCRs in mammalian cells, purify via a variety of affinity tags, and solubilize the receptors in detergent micelles. We have developed a systematic method for identifying detergents optimal for activity and stability of the receptor, and for assessing the contribution of protein-detergent interactions to receptor stability. In collaboration with the Kaler and Lenhoff groups in Chemical Engineering, we are developing robust methods to identify detergent effects crystallization conditions, based on measuring osmotic virial coefficients. In collaboration with the Anne Robinson group in Chemical Engineering, we are developing improved methods to produce active receptors from E. coli inclusion bodies or inactive states, using a novel refolding scheme.
Design and Engineering of GPCR Analogs for Drug Discovery
We believe that many GPCRs contain a core ligand binding domain, that can be expressed as an independent protein chain, and used in drug screening or development applications. My previous research showed that a four helix variant of the b2 adrenergic receptor retains ligand binding affinity, and specificity mirroring that of the native receptor. We now seek to produce similar GPCR analogs for several members of the adenosine and adrenergic receptor subfamilies. Using a variety of methods, including rational mutagenesis, directed evolution, and generation of fusion proteins, we intend to generate soluble receptor analogs that are suitable for use in high-throughput screening applications. For other GPCR subfamilies, including the glucagon family (serpins), and FSH and related receptors, ligand binding determinants are concentrated in large N-terminal regions, which we can express as active, soluble receptor fragments. In both cases, these analogs will have the advantage of being more tractable targets for structural biology, which will facilitate structure-based design and lead optimization.
Determinants of GPCR Stability and Assembly
Understanding the principle of stability, folding, and assembly for GPCRs can have a major impact on our ability to produce and characterize these receptors. We use spectroscopic methods, including fluorescence and circular dichroism, to monitor conformation of receptors, analogs, and site-directed mutants. We have shown that folding of at least one receptor, b2AR, proceeds via an intermediate state; this finding led us to develop the four-helix analog described above. The receptor can fold via an alternate pathway that leads to an inactive state, whose conformation closely resembles the native state. Separating these two conformations is obviously critical for structural and functional studies. The branch between active and inactive folding pathways appears to be controlled by interactions between cysteine residues in the transmembrane domains. We are investigating whether other receptors fold via this pathway, and using this information in our development of robust expression and refolding schemes.
GPCR信號通路圖
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作者簡介：
Hong-Sheng Li, Ph.D.
Academic Role: Assistant Professor
Faculty Appointment(s) In:
Neurobiology
Other Affiliation(s):
Program in Neuroscience
Neuronal signal transduction and degeneration in the fly eye
We are interested in neuronal signal transduction events that involve Ca2+, and their consequences in gene expression and neurodegeneration. Using the fly eye as the major experimental system, our research is currently focused on the following three related topics:
1. GPCR-operated ion channels
G-protein coupled receptors (GPCR) initiate signal transduction in several peripheral sensations, such as vision, olfaction, and some taste sensations. In the central nervous system, they serve as receptors for numerous neurotransmitters and neuropeptides, whereby they modulate synaptic transmission and neuronal excitability. Our previous work indicates that a family of Ca2+-permeable ion channels, TRPC, could be the major membrane effectors of GPCR in central neurons. The stimulation of TRPC by GPCR is mostly mediated by phospholipase C (PLC). However, the steps that directly lead to channel activation and regulation are still unclear.
The fly ion channel transient receptor potential (TRP) is the first identified member of the TRPC family. It is highly enriched in the rhabdomere, the light-sensing organelle in fly photoreceptor neurons, and stimulated by the G-protein coupled light receptor rhodopsin. Since TRP channel activities can be easily detected by electroretinogram recording, and fly genetic tools are powerful, we have been using the photoreceptor neuron as an in vivo system for the TRPC study. In a recent mutagenesis screen, we isolated two fly mutant lines that appeared to be defective in TRP channel deactivation. We are now in the process of identifying the mutated genes.
2. Ca2+/Camodulin-regulated gene expression
Most TRPC channels are permeable to Ca2+, which is a major second messenger that GPCR utilize to regulate neuronal activities. Ca2+ signals are capable of producing long-term effects through changing gene expression. By the mediation of CaM Kinases or calcineurin, Ca2+/Camodulin activate several transcription factors, including CREB, SRF, NFAT and NFκB. Recent studies in plants found that a new group of transcription factors, CAMTA, could be activated directly by Ca2+/Camodulin, and thus may respond to Ca2+ signals at much higher speed. We have isolated a fly line with mutations in the CAMTA gene. Our preliminary data indicate that the presence of a target gene of CAMTA might be required for the rapid deactivation of TRP channels. We have been charactering both fly and human CAMTA proteins and identifying their target genes.
3. Degeneration in photoreceptor neurons
Neuronal activities, especially those involving Ca2+, have a direct impact on cell viability, and usually play a role in neurodegeneration. For example, both unregulated TRP channel opening and low channel activities due to rhodopsin, PLC or TRP gene mutation cause fly rhabdomeral degeneration. In addition, neurodegeneration could result from defects in structural genes or metabolic enzymes. In a mutagenesis screen using a morphological assay, we isolated three new fly mutants undergoing rhabdomeral degeneration. Once the mutant genes are identified, we will test them for interactions with known degeneration genes. Since the fly photoreceptor neuron is a model system for both retinal and central neuron degeneration, our research findings may ultimately help to elucidate the mechanisms of neurodegeneration in humans.
